Throughout this application, various publications are referenced. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citations for the references may be found listed immediately preceding the claims.
Antibodies specific for tumor-associated antigens can provide effective vehicles for in vivo delivery of agents, such as radionuclides, for detection or therapy of cancer. The potential utility of cancer-targeting antibodies can be improved by protein engineering approaches, which can be used to modify characteristics such as affinity, immunogenicity, and pharmacokinetic properties. In particular, recombinant antibody fragments have been produced with favorable characteristics, including retention of high affinity for target antigen, rapid, high level accumulation in xenografts in murine models, and quick clearance from the circulation, resulting in high tumor:normal activity ratios. Furthermore, because antibody fragments do not persist in the circulation, they are less likely to be immunogenic than intact murine or even chimeric antibodies. With the advent of humanized and human antibodies, the issue of immunogenicity of recombinant antibodies is rapidly diminishing.
Recombinant fragments such as diabodies (e.g., 55 kDa dimers of single-chain Fv fragments, which self-assemble in a cross-paired fashion as described by Holliger, et al., 1993) or minibodies (e.g., 80 kDa scFv-CH3 fusion proteins as described by Hu et al., 1996)) have shown promise as in vivo imaging agents in preclinical studies when radiolabeled with single-photon emitting radionuclides such as In-111 or 1-123, or positron emitters such as Cu-64 or 1-124 for positron emission tomography (Sundaresan et al., In press; Wu et al. 2000). Targeting and imaging of 1-123 radiolabeled single-chain Fv (scFv, 27 kDa) fragments has been demonstrated clinically, although the size and monovalency of scFv's may limit their utility (Begent, et al., 1996). Recent clinical imaging studies using I-123 radiolabled diabodies appear promising (Santimaria et al. 2003).
Most current antibody radiolabeling approaches involve conjugation to random sites on the surface of the protein. For example, standard radioiodination methods result in modification of random surface tyrosine residues. Many antibodies are highly susceptible to inactivation following iodination, presumably due to modification of key tyrosines in or near the binding site. (Nikula et al., 1995; Olafsen et al., 1996). Chemical modification of lysines located in or near the antigen-binding site could also potentially interfere with binding through sterical hindrance if a bulky group is added (Benhar et al., 1994; Olafsen et al., 1995). Alternative iodination approaches or radiometal labeling through conjugation of bifunctional chelates direct modifications to ε-amino groups of lysine residues, again randomly located on the surface of antibodies. The issue of inactivation following radiolabeling becomes more pressing as one moves to smaller and smaller antibody fragments, if equal reactivity is assumed, because the binding site(s) represent a larger proportion of the protein surface, and fewer “safe” sites for conjugation are available.
Site-specific radiolabeling approaches provide a means for both directing chemical modification to specific sites on a protein, located away from the binding site, and for controlling the stoichiometry of the reaction. Several strategies capitalize on naturally occurring moieties or structures on antibodies that can be targeted chemically. For example, the carbohydrate found on constant domains of immunoglobulins can be oxidized and conjugated with bifunctional chelates for radiometal labeling. (Rodwell, et al., 1993). In one instance, an unusual carbohydrate moiety occurring on a hypervariable loop of a kappa light chain was modified for site-specific chelation and radiometal labeling (Leung, et al., 1995). Others have exploited selective reduction of interchain disulfide bridges to enable modification using thiol-specific reagents. C-terminal cys residues on antibody Fab or Fab′ fragments have been used for direct labeling using 99mTc (Behr, et al., 1995; Verhaar, et al., 1996). Novel approaches include the identification of a purine binding site in antibody Fv fragments, allowing specific photoaffinity labeling (Rajagopalan, et al., 1996).
More recently, genetic engineering approaches have been used to introduce specific sites for modification or radiolabeling of proteins and antibodies. Building on the above-mentioned work, glycosylation sites have been engineered into proteins to provide novel carbohydrate targets for chemical modification (Leung, et al., 1995; Qu, et al., 1998). The six-histidine tail commonly appended to recombinant proteins to provide a purification tag has been used in a novel 99mTc labeling method (Waibel, et al., 1999). Alternatively, a popular strategy has been to use site-directed mutagenesis to place cys residues on the surface of proteins to provide reactive sulfhydryl groups. This approach has been implemented by numerous groups to allow site-specific labeling of antibodies (Lyons, et al. 1990; Stimmel, et al., 2000) and other proteins (Haran, et al., 1992; Kreitman, et al., 1994).
Introduction of cys residues into engineered antibody fragments also has been used for stabilization or multimerization purposes. For example, introduction of strategically placed cys residues in the interface between the VH and VL domains of antibody Fv fragments has allowed covalent linkage and stabilization of these fragments (disulfide-stabilized Fv, or dsFv) (Glockshuber, et al., 1990; Webber, et al., 1995). Fitzgerald et al. described a disulfide bonded diabody in which cysteine residues were introduced into the VL/VH interface for stability and demonstrated its utility for fluorescent imaging of tumors (Fitzgerald, et al., 1997). Others have appended cys residues to the C-termini of single-chain Fv fragments (scFv, formed by fusing VH and VL with a synthetic peptide linker) to allow multimerization into scFv′2 fragments (Adams, et al., 1993; Kipriyanov, et al., 1995).
We have previously produced an anti-carcinoembryonic antigen (anti-CEA) diabody from the murine anti-CEA T84.66 antibody by joining VL—eight amino acid linker—VH. Tumor targeting, imaging, and biodistribution studies of a radiolabeled (at random sites on the protein) anti-CEA diabody demonstrated rapid tumor uptake, fast clearance from the circulation, and favorable properties for use as an imaging agent, when evaluated in nude mice bearing LS14T xenografts (Wu, et al., 1999; Yazaki et al., 2001b).
There remains a need in the art, however, for a stable, in vivo delivery vehicle that can be modified readily in specific locations without affecting the ability of the vehicle to specifically target cells of interest. There is also a continuing need for better in vitro detection methods. The invention provides a system for adding site-specific functional groups to antibody fragments that do not interfere with target binding by said fragment.